【作者】 陈克研；

【导师】 高丰；

【作者基本信息】 吉林大学 ， 基础兽医学， 2011， 博士

【摘要】 猪血凝性脑脊髓炎是由血凝性脑脊髓炎病毒(hemagglutinating encephalomyelitis virus, HEV)引起仔猪的一种急性、接触性传染病。HEV属于冠状病毒属成员,其主要侵害1-3周龄的仔猪,临床上以仔猪呕吐、衰竭和明显的神经症状为主要特征,死亡率达20-100%。1962年,该病原从加拿大患脑脊髓炎的哺乳仔猪体内首次分离获得。1972年,比利时人Pensaert MB从暴发“呕吐和衰竭”症状,而未出现“脑脊髓炎”神经症状的死亡猪扁桃体中分离获得HEV-VW572毒株。以后,世界上许多国家均有该病的报道,尤其以日本、加拿大等国家危害比较严重。血清学调查显示,猪感染HEV非常普遍,且呈世界性分布,大部分被感染的猪处于亚临床状态,一旦发病,将给养猪业造成巨大的经济损失。目前,常规的HEV检测技术主要有病毒分离鉴定、RT-PCR、巢式PCR、血凝/血凝抑制试验(HA/HI)、血清中和试验(VN)等方法。但这些检测方法大多需要专业的技术人员、特定的操作环境以及一些特殊的仪器设备,不适合基层现场检验工作的需要,满足不了该病暴发流行时紧急应对的要求,所以在一定程度上限制了其在兽医临床诊断中的应用。因此,开发一种操作简单、使用方便快捷的HEV检测方法对于基层兽医工作者十分必要。近年来,随着单克隆抗体(McAb)技术的发展与成熟,其在分子生物学、免疫学、生物化学、遗传学等许多领域得到了广泛的应用,许多疾病诊断和鉴别诊断方法的建立都与McAb有关。本研究应用蔗糖密度梯度离心法纯化HEV,免疫Balb/c小鼠,取小鼠脾细胞与SP2/0骨髓瘤细胞进行融合,用间接ELISA筛选杂交瘤细胞,获得4株稳定分泌抗HEV的单克隆抗体,分别命名为2H2、2A1、1E2、4D4。经鉴定,4株McAb的ELISA效价在1：12800～1：51200之间；亚类鉴定证实,除1E2为IgG2a外其余3株单抗均为IgGl;Western blot分析表明,2H2、1E2和4D4能识别HEV的血凝素-酯酶蛋白(HE),2Al可识别纤突蛋白(S)；经间接ELISA及间接免疫荧光鉴定,4株单抗均能与HEV结合,其特异性良好；用单抗2A1和2H2建立检测HEV的双抗体夹心ELISA方法,最低可检测3.75μg/mL的病毒,且与猪传染性胃肠炎病毒(TGEV)、猪流行性腹泻病毒(PEDV)、猪伪狂犬病毒(PRV)、猪瘟病毒(HCV)及牛冠状病毒(BCV)等无交叉反应,说明该方法可用于HEV的检测。胶体金免疫层析技术(Gold Immunochromtographic assay, GICA)是将免疫胶体金技术(Immunogold labeling technique)与层析法结合,以胶体金作为标记物,应用于抗原与抗体之间反应的一种新型检测技术。将McAb与GICA结合制备的胶体金检测试纸条具有操作简单快速、检测结果清楚易于判断、特异性强、敏感性高、无需仪器设备或只需简单的仪器等优点,因此非常适于发病现场、门诊以及实验条件不具备的场所使用,在医学和动物医学检疫、检验等方面已被广泛应用。本实验在成功制备HEV单克隆抗体的基础上,结合胶体金免疫层析技术研制检测HEV的胶体金免疫层析试纸条。首先应用柠檬酸三钠还原法制备胶体金溶液,经电镜观察胶体金颗粒大小在30nm左右；通过梯度法确定McAb与胶体金结合的最佳pH值为8.5,最佳结合浓度为37μg/mL;将制备好的金标单抗包被玻璃纤维膜,另一株McAb及羊抗小鼠IgG分别喷点硝酸纤维膜(NC膜)检测线和质控线,组装试纸条。用该试纸条检测不同浓度的HEV,结果最低可检测30μg/mL的病毒；特异性实验结果表明该试纸条在检测TGEV.PEDV.PRV、HCV.BCV及MHV等病毒时不发生交叉反应；分别用试纸条.RT-PCR及ELISA方法对50份临床样本做平行检测,结果试纸条与RT-PCR和ELISA的符合率为98%,其Kappa值为0.956,说明本实验研制的试纸条可用于临床HEV的检测。应用胶体金免疫层析技术研制检测HEV血清抗体的胶体金免疫层析试纸条,首先将兔抗猪IgG标记胶体金包被玻璃纤维膜,然后用喷点仪将纯化的HEV和羊抗兔IgG分别喷点于NC膜的检测线和质控线,组装成试纸条。用该试纸条分别检测HEV、TGEV、PEDV、PRV、HCV及BCV的阳性血清并用其检测不同HI抗体效价的HEV阳性血清,结果试纸条仅对HEV阳性血清检测时,在检测线及质控线出现清晰的红色条带,且最低可检测HI效价为21的HEV阳性血清,说明试纸条具有很好的特异性和敏感性；用同一批次和不同批次的试纸条检测HEV阳性血清,结果试纸条批内重复及批间重复差异不大,变异系数为0.82%,证明试纸条重复性较好；保存期试验证明该试纸条4℃保存12个月,其特异性和敏感性均未发生变化,说明试纸条具有很好的稳定性；分别用试纸条、ELISA及HI试验对50份临床血清样本进行检测,评估试纸条与二者的相关性。结果试纸条与ELISA的符合率为98%, Kappa值为0.953；与HI试验的符合率为96%,Kappa值为0.911,说明试纸条可代替ELISA和HI试验用于临床血清样本检测。应用本实验室研制的试纸条对吉林省及辽宁省部分地区进行HEV的血清抗体调查,结果672份血清样本中,有334份呈阳性,总阳性率为49.7%,提示被检测地区的猪群中普遍存在HEV感染。目前,HEV的暴发呈上升趋势,且未见有关预防或治疗HEV的特效药物或疫苗的研究报道,仅在国外部分国家应用“亚感染”进行预防,即在母猪临产前一个月与病猪接触或喷雾或肌肉注射培养的弱毒,使母猪获得免疫,进而通过初乳中的抗体保护后代仔猪,使其不表现临床症状,而这些猪大多呈亚临床感染,且此种免疫方法存在着散毒的危险。因此,研制高效安全的疫苗对HEV的防治有重要的现实意义。本试验选用HEV-67N标准毒株作为疫苗研制的候选毒株,通过优化体外培养条件,确定HEV-67N在PK-15细胞上培养传代,并建立PK-15细胞的原始种子细胞库(123～126代细胞)、基础种子细胞库(127～136代细胞)和工作种子细胞库(137～141代细胞)；同时分别建立HEV-67N原始种子病毒库、基础种子病毒库和工作种子病毒库；随机抽取细胞库及病毒库中的细胞和病毒,通过镜下观察、无菌检验、支原体检测及核酸分析等检测均符合“兽用生物制品质量标准”；然后在筛选出优良佐剂的基础上,取工作种子病毒库的病毒液,经4%o甲醛灭活后,按《中华人民共和国兽用生物制品质量标准》规定,将氢氧化铝胶佐剂和灭活的病毒按1∶9比例混匀,制备HEV细胞培养灭活疫苗。随机抽取实验室制备的疫苗以不同剂量接种Balb/c小鼠和怀孕母猪,结果该疫苗除了可以诱导良好的特异性免疫应答外,不产生任何临床副作用,说明本疫苗对小鼠和怀孕母猪是安全有效的；对注射疫苗后产生不同抗体效价的Balb/c小鼠进行脑内攻毒,结果当小鼠HI抗体效价≥25时,其保护率可达80%以上,且HI抗体效价滴度与免疫攻毒试验间存在良好的平行关系,可代替常规的攻毒保护率试验；将HEV灭活疫苗免疫Balb/c小鼠,结果小鼠经3次免疫后,HI抗体效价最高达29,进而通过血清亚类鉴定、淋巴细胞增殖实验、细胞因子检测等方法证实,该疫苗可刺激小鼠产生特异性的体液免疫应答,且以Th2型免疫应答为主；最后对疫苗的保存期进行评定,疫苗在2～8℃和室温(13℃～28℃)分别保存至12个月和4个月时,其物理性状及免疫效力未见变化,这与灭活疫苗稳定、便于保存运输的特点基本一致。由此可见,本实验制备的HEV灭活疫苗可安全有效的刺激机体产生特异性免疫应答,仔猪可通过经疫苗免疫的怀孕母猪产生HEV抗体,从而达到预防该病的目的,该疫苗的研制对HEV的防治有重要的现实意义。更多还原

【Abstract】 Porcine hemagglutinating encephalomyelitis is an acute, highly contagious disease in piglets caused by the hemagglutinating encephalomyelitis virus (HEV). HEV belongs to the family of Coronaviridae, which included encephalomyelitis and Vomiting and wasting disease clinically type and mainly infects 1-to 3-week-old piglet causes vomiting, exhaustion, and neurological signs. The mortality rate varies from 20% to 100%. In 1962. the pathogen was isolated for the first time from suckling piglets suffering from encephalomyelitis in Canada. In 1972, the PHEV-VW572 strain was isolated from the tonsils of pigs that exhibited only vomiting and exhaustion symptoms, but no neurological signs. Serological surveys revealed that HEV infections in pigs are very common worldwide, most of the infected pigs in a sub-clinical state, once the disease, will cause huge economic losses. At present, various laboratory methods are available for the detection and surveillance of HEV. including virus isolation and identification, RT-PCR, nested PCR, hemagglutination and hemagglutination inhibition (HA and HI,respectively) tests, the enzyme-linked immunosorbent assay (ELISA). and the virus neutralization (VN) test. However, these detection methods are laborious, time-consuming, and require laboratory operations or special equipment, which makes these methods unsuitable for on-site inspection. As such, the current methods would not be useful for managing an outbreak of the disease. Therefore, it was necessary to develop a sensitive, specific, and easily performed detection method for on-site detection of HEV or HEV antibodies in order to increase the surveillance of HEV infections.In recent years, with the monoclonal antibody (McAb) technology development and maturation, molecular biology, immunology, bacteriology, virology, biochemistry, genetics and many other fields have been widely used. Many diseases, diagnosis and differential diagnosis were established with the McAb.In this study, Balb/c mice were immunized subcutaneously with HEV purified by sucrose density gradient centrifugation, their splenic mononuclear cells were isolated and fused with murine myeloma cells (SP2/0). The hybridomas were generated through the selection of HAT medium and screened using Enzyme-linked immunosorbent assay (ELISA) and access to four single-secreting anti-HEV McAb, named 2H2,2A1,1E2,4D4. Identified,4 McAb ELISA titers between 1:12800 and 1:51200; subgroup identification,1E2 for the IgG2a,2H2、2A1 and 4D4 for the IgG1;Western blot analysis showed that 2H2,1E2 and 4D4 to recognize HEV hemagglutinin-esterase protein (HE),2A1 may recognize spike protein (S). Development of the sandwich ELISA with McAb 2A1 and 2H2 for detection HEV, the minimum could be detected 3.75μg/mL for HEV, and infection with the transmissible gastro-enteritis virus (TGEV), porcine Epidemic diarrhea (PEDV), porcine pseudorabies virus (PRV), Hogcholera virus (HCV) and bovine coronavirus (BCV) and so no cross reaction, wihich shows that the sandwich ELISA can be used for HEV detection.Colloidal gold immunochromatographic assay (GICA) is a solid-phase immunoassay developed in the 1980s that combines the techniques of colloidal gold labeling, chromatographic analysis, immunodetection and other methods. Because of GICA’s convenience and speed as well as its specificity and sensitivity and ability to be used without instruments or with only a simple instrument, it is suitable for clinical diagnosis and drug testing purposes and can be used anywhere.In this study, an immunochromatographic strip with high sensitivity and specificity was successfully developed for the detection of HEV. combining McAb and GICA.The colloidal gold particles were consistent in size and uniformly distributed, with a mean diameter of about 30nm when observed under a transmission electron microscope; monoclonal antibodies by gradient method to determine the best combination of colloidal gold pH 8.5, the best combination of a concentration of 37μg/mL. The colloidal gold-labeled MAb 2H2 solution was dispensed onto glass fiber paper at a speed of 50μl per cm using an XYZ3000 Dispense Workstation, and the MAb 2A1 or the goat anti-mouse antibody was dispensed at the test or the control line on the NC membrane using XYZ-3000; the sample pad, pretreated conjugate pad. NC membrane, and absorbent pads were glued together on a support board and assembled into a test strip.The immunochromatographic strip was capable of specifically detecting HEV with 30μg/mL within 10 min. Storage of the strips at room temperature for 6 months or at 4℃for 12 months did not change their sensitivity or specificity. Using RT-PCR and ELISA as reference test, the excellent agreement (98%; kappa= 0.956) between the results obtained by RT-PCR or ELISA and the immunochromatographic strips. Based on its high specificity, sensitivity, and stability, the immunochromatographic strip would be suitable for on-site detection of HEV for surveillance and epidemiological purposes.An immunochromatographic strip was developed for the detection of an antibody against HEV. Colloidal gold-labeled rabbit anti-pig immunoglobulin G (IgG) was used as the detection reagent, and the HEV recombinant antigens and goat anti-rabbit IgG were coated on the prototype strip and the control lines, respectively. The immunochromatographic strip was capable of specifically detecting HEV antibodies in serum with a HI titer of 2 within 10 min. Storage of the strips at room temperature for 6 months or at 4℃for 12 months did not change their sensitivity and specificity. Using HI as a reference test, there was a strong agreement between the results obtained by HI and the immunochromatographic strips (kappa=0.911). Additionally, there was a strong agreement between ELISA and immunochromatographic strips (kappa=0.953). When the immunochromatographic strip was used for serological diagnosis of 672 serum samples in the Jilin and Liaoning province in China, the seropositivity ranged from 42.08% in Jinzhou District to 65.33% in Baishan District. Based on the high specificity, sensitivity, and stability of the immunochromatographic strip, it would be suitable for on-site detection of HEV antibodies in order to monitor HEV infections in an animal population.Currently, there is no effect on the prevention or treatment of HEV drugs or vaccines has been reported, applied only in some countries abroad, "subclinical infection" of prevention, that is, one month before labor in sows contact with sick, spray or intramuscular injection training the attenuated HEV. When the sow immunitied, piglets gain the antibodies through colostrum, but it does not show clinical symptoms, most of these pigs showed a subclinical infection, and this immunological method exist clinical infection hidden danger. Therefore, the development of efficiently and safely vaccine against HEV prevention has important practical significance. In this study, based on HEV-67N strain as a vaccine candidate strains developed by optimizing the in vitro culture conditions to determine the HEV-67N PK-15 cells in culture and passage, using the nutrient medium with 0.4% BSA; establishment of PK-15 cells of the original seed cell banks (123～126 cells), based on the seed cell bank (127～136 cells) and the working seed cell bank (137～141 cells); the same time establish HEV-67N, respectively, the original seed virus database. foundation seed and working seed virus database virus database; random sample of the virus in the cell library and PK-15 cells and HEV-67N by microscopy, sterility test, mycoplasma detection and analysis of nucleic acids which adhere to "quality standards for veterinary biologics." In the adjuvant selected on the basis of quality, taking the virus working seed virus database solution, inactivated by 4‰formaldehyde, By "People’s Republic of Veterinary Biological Products Quality Standards " provides, the aluminum hydroxide gel adjuvant and inactivated virus at 1:9 ratio of mixing, preparation HEV cell culture inactivated vaccine. Randomly selected the vaccine which prepared in labs Vaccination to Balb/cmice and pregnant sows, the results show that favourable specific immune response induced by the vaccine. The vaccine is also safe an effective to mice and pregnant sows. There is no adverse reaction happened on mice and sows after injecting the vaccine. On post-vaccination antibody titers have different Balb/c mice brain attack drugs, the protection rate is more than 80% when the mice’s HI antibody titers≥2(?).There is satisfactory parallel relationship between HI antibody titers and immune attack drug test. It can replace convetional attack protection rate test. HEV inactivated vaccine inoculated Balb/c mice, HI antibody titers of mice up to 29 after the third immunization. The immune mice could produce specific humoral immune response certificated by serum sub-categories, lymphocyte proliferation, cytokines and other methods. The main of immune response is Th2 type. Finally, the preservation of the vaccines evaluated. The physical properties and immune effect of the vaccine was no change when the vaccine stored at 2-8℃for 12 months and room temperature (13℃-28℃) for 4 months.It basically the same characteristics as inactivated vaccine stability, ease of transportation and conservation. Thus, in this study, the body could produce specific immune response by the prepared HEV vaccine safely and efectively. Piglets generated the antibodies from the immunization of pregnant sows to achieve the purpose of preventing the disease.The vaccine has important practical significance to prevention of HEV更多还原